Construction and analysis of a library for random insertional mutagenesis in Streptococcus pneumoniae: Use for recovery of mutants defective in genetic transformation and for identification of essential genes

To explore the use of insertion-duplication mutagenesis (IDM) as a random g ene disruption mutagenesis tool for genomic analysis of Streptococcus pneum oniae, a large mutagenic library of chimeric plasmids with 300-bp inserts w as constructed. The library was large enough to produce 60,000 independent plasmid clones in Escherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scatter ed widely over the genome; 80% of these plasmids had 240- to 360-bp inserts , and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evalua te the randomness of mutagenesis during recombination and to test the usefu lness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a la rge pneumococcal transformant library derived from 20,000 mutagenic plasmid s. After we screened the mutants exhaustively for transformation defects, 1 14 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as w ell as a new gene with high similarity to the Bacillus subtilis competence gene comFA, Mapping of the mutation sites at these competence loci showed t hat the mutagenesis was highly random, with no apparent hot spots. The reco very of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is usefu l for finding various types of nonessential genes throughout the genome. Si nce a promoterless lacZ reporter vector was used for the construction of th e mutagenic plasmid library, it also serves as a random transcriptional fus ion library. Finally, use of a valuable feature of IDM, directed gene targe ting, also showed that essential genes, which can be targets for new drug d esigns, could be identified by simple sequencing and transformation reactio ns. We estimate that the IDM library used in this study could readily achie ve about 90% genome coverage.